Radiocarbon ages of calcium oxalate that occurs naturally on rock surfaces have been used recently in archaeological and paleoenvironmental studies. Oxalate rock coatings are found globally, with most appearing to be residues from epilithic lichens. To explore the source(s) of carbon used by these organisms for the production of oxalate we measured the natural abundances of 14C and 13C in 5 oxalate-producing lichen species, 3 growing on limestone in southwestern Texas and 2 on sandstone in Arkansas. We also examined the distribution of the isotopes between the calcium oxalate and lichen tissues by separating these components and measuring the 13C/C independently. The results demonstrate that the limestone species were slightly enriched in 14C, by 1.7%, relative to the sandstone species, which suggests that “dead” carbon from the limestone substrate does not constitute a significant source of carbon for the production of oxalate. The calcium oxalate produced by the lichens is also enriched in 13C by 6.5% compared to the lichen tissues, demonstrating that there is a large carbon isotope discrimination during oxalate biosynthesis. These results support the reliability of 14C ages of calcium oxalate rock coatings used for archaeological and paleoclimate studies.